This invention relates to the treatment of HIV infection. More particularly, this invention relates to synthetic modified antisense oligonucleotides and pharmaceutical compositions containing such oligonucleotides and to methods of inhibiting HIV replication and treating HIV infection using such oligonucleotides.
Human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2), formerly called human T-cell leukemia lymphotropic virus-type III (HTLV-III), are believed to be the etiological agents of acquired immune deficiency syndrome (AIDS). HIV is part of the Retroviridaie family, the members of which contain an RNA genome and reverse transcriptase activity. During their growth cycle, retroviruses copy their RNA into proviral DNA. The proviral DNA is able to integrate into the chromosomal DNA of the host cell where it uses the transcriptional and translational machinery of the host to express viral RNA and proteins. Viruses are released from the cell by budding from the cytoplasmic membrane. In the case of HIV-1 and HIV-2, viral replication results in the death of helper T-cell host cells, which leads to a state of severe immunodeficiency, to the development of various malignancies and opportunistic infections, and ultimately to the death of the infected organism.
The incidence of AIDS has risen to epidemic proportions in many countries without the development of preventative treatments or therapies which are successful in the long term. Those few therapeutic agents which have been prescribed, such as the nucleoside analogs 3′-azido-3′-deoxythymidine (AZT), dideoxyinosine (ddI), and dideoxycytosine (ddC), and various protease inhibitors have met with limited success. This has been in part because of the cytotoxicity of these agents. In addition, some viruses escape due to mutations that render them insensitive to these agents and the difficulty of antiviral action due to the ability of the virus to integrate into the host's genome. Thus, there is a long felt need for more effective therapeutic agents and preventative therapies for AIDS.
More recently new chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression. These agents, called antisense oligonucleotides, bind to a target singe-stranded nucleic acid molecules according to the Watson-Crick or the Hoogstein rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal translation or transcription; in the case of an mRNA target, by triggering the enzymatic destruction of the message by RNase H; or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Antisense oligodeoxynucleotides have been designed to specifically inhibit the expression of HIV-1 and other viruses (see, e.g., Agrawal (1992) Trends in Biotechnology 10:152–158; Agrawal et al. in Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds.) Raven Press Ltd., New York (1992) pp. 273–283); Matsukura et al. in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wiley-Liss, Inc. (1992) pp. 159–1798); and Agrawal (1991) in Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, (Wickstron, ed.) Liss, New York, pp. 145–148). For example, it has been shown that antisense oligonucleotides having phosphodiester internucleoside bonds and sequences complementary to portions of genomic HIV-1 RNA inhibit viral replication in early infected cells (Zamecnik et al. (1986) Proc. Acid. Sci. USA 83:4143–4147; Goodchild et al. (1988) Proc. Natl. Acad. Sci USA 85:5507–5511).
However, these phosphodiester-linked molecules are less able to inhibit viral replication in chronically infected cells (Agrawal et al. (1989) Proc. Natl. Acad. Sci USA 86:7790–7794), mainly because of their nuclease susceptibility (Wickstrom (1986) J. Biochem. Biophys. Meth. 13:97–102). Therefore, chemically modified, nuclease-resistant analogs have been developed which are effective in inhibiting HIV-1 replication in tissue cultures (see, Sarin et al. (1988) Proc. Natl Acad. Sci. USA 85:7448–7451; Agrawal et al. (1988) Proc. Natl Acad. Sci USA 85:7079–7083; Matsukura et al. (1988) Gene 72:343–347). These analogs include oligonucleotides with nuclease-resistant phosphorothioate internucleotide linkages shown to inhibit HIV-1 replication in both acute infection (U.S. Ser. No. 08/309,823; Agrawal et al. (1989) Proc. Natl. Acad. Sci USA 86:7790–7794) and in chronically infected cell lines (Agrawal et al. (1991) in Gene Regulation: Biology of Antisense RNA, eds. Erickson et al. (Raven Press, New York), pp. 273–284; Vickers et al. (1991) Nucleic Acids Res. 19:3359–3368; Matsukura et al. (1989) Proc. Natl Acad. Sci. 86:4244–4248; Agrawal et al. (1988) Proc. Natl Acad. Sci USA 85:7079–7083).
However, some phosphorothioate-linked oligonucleotides that have “GC-rich” nucleotide sequences have been found to evoke immunostimmulatory responses in the organisms to whom they have been administered. For example, Kniep et al. (Nature (1995) 374:546–549) discloses that oligonucleotides containing the CG dinucleotide flanked by certain other sequences have a mitogenic and other side effects.
Thus, there still remains a need for a more effective anti-HIV oligonucleotide having therapeutic effects that are accompanied by fewer side effects, e.g., little cellular toxicity and reduced immunostimmulatory response.